Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand

12(S)-hydroxyheptadeca-5Z,8 E,10 E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A(2) (TxA(2)) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B 4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB(2), a stable metabolite of TxA(2). TxB(2) production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD(2) and PGE(2), possibly resulting from the shunting of PGH(2) into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB(2) production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB(2) and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB(2). These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo.